Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for analyzing biophysical properties of gap junctions. The main advantage of our technique is to use a high-side current measuring approach that is appropriate for double site voltage clamps. The procedure will be demonstrated by Yuan Shui, a post-doctoral fellow from my laboratory.
As soon as 70 to 80%of the oocytes are defoliculated, decant the enzyme solution by gently tilting the tube. Wash the oocytes five times by filling the tube with ND96 solution and then decanting the solution. After the final wash, transfer the oocytes to a 60-millimeter Petri dish containing ND96 solution.
Use a clean glass Pasteur pipette to pick and transfer large, healthy-looking oocytes to a 60-millimeter Petri dish containing ND96 solution supplemented with sodium pyruvate and penicillin streptomycin. Prepare an oocyte injection dish by gluing a small piece of nylon mesh to the bottom of a 35-millimeter Petri dish. Fill the oocyte injection dish approximately halfway with ND96 solution supplemented with pyruvate and penicillin streptomycin.
Then, place 25 to 30 oocytes in rows inside the Petri dish. Back fill an injection micropipette with lightweight mineral oil and insert it into the micropipette holder of the injector. Transfer the cRNA from one of the stored aliquots to the inside surface of a Petri dish cover or bottom by pipetting.
Press the fill button in the injector controller to aspirate the cRNA droplet into the tip of the micropipette. Then, press the inject button to inject the cRNA. Transfer the injected oocytes to a new Petri dish containing ND96 solution supplemented with pyruvate and penicillin streptomycin.
Keep them inside the environmental chamber at 15 to 18 degrees Celsius for one to three days. Replace the solution daily by transferring the oocytes to a new Petri dish. Use two fine tweezers to gently peel off the transparent villin membrane that wraps the oocyte.
Then, place two oocytes per well in an oocyte pairing chamber containing the ND96 solution. Connect the differential voltage probe and the current cables to the model cell. Then, connect the red and black sockets to the differential voltage probe with the included jumper and connect either leg of the jumper to either pin in the model cell.
Connect the ground wire in the model cell to the cable from the grounds circuit socket of the amplifier. Turn the VM offset and VE offset knobs to zero the membrane voltage and membrane current meters. Switch the clamp from off to either fast or slow and turn the gain dial clockwise to a level that allows proper voltage clamp to zero the voltage electrode and bath electrode meters of the amplifier.
Run a simple acquisition protocol containing a few voltage steps to confirm that the voltage displayed in the membrane voltage meter changes according to the acquisition protocol, and that voltage and current traces are displayed properly in Clampex. Place a Petri dish containing the paired oocytes into the Petri dish receptacle of the recording platform. Select a pair of oocytes and rotate the Petri dish if necessary, so that the two oocytes are in the left and right direction.
Lock the stage in position by its magnetic base. Place a reference electrode near the edge of the Petri dish towards the user side. Then, connect the reference electrode to the black socket in only one of the two differential voltage probes.
Take a pair of glass micropipettes, break off a bit of the tip with a diamond scriber, and smooth the tip edge by fire polishing. Hold the micropipette above the flame of an alcohol burner and bend it to a smooth angle at approximately one centimeter away from the tip. Back fill the glass pipette completely with ND96 solution and then insert it into a pre-filled microelectrode holder.
Ensure that no air bubbles are present in the system. Insert the two-millimeter pin of the microelectrode holder into the two-millimeter socket of a differential voltage electrode connection wire. Clamp the two-millimeter socket on a magnetically-based clamper and aim the tip of the differential voltage electrode toward one of the two oocytes.
Insert the one-millimeter pin of the differential voltage electrode connection wire into the red socket of the differential voltage probe on the same side. Prepare the voltage and current electrodes from the glass capillaries and back fill them with potassium chloride solution. Then, insert the electrodes into the electrode holders provided with the amplifiers.
Lower the electrodes into the bath solution and turn the VM offset and VE offset dials to zero the membrane voltage and membrane current meters. Check the electrode resistance by pressing VM electrode test and VE electrode test. Insert the current and voltage electrodes into the oocytes and observe the negative membrane potentials.
Next, in the clamp section of the amplifier, confirm that DC gain is at the in position. Turn the gain knob clockwise to a level that allows proper voltage clamp and turn the clamp switch from off to fast. Finally, run an acquisition protocol.
A diagram of the oocyte experiment is shown here. Negative and positive membrane voltage steps are applied to oocyte 1 from a holding membrane voltage of minus 30 millivolts, whereas oocyte 2 is held at a constant membrane voltage of minus 30 millivolts. The trans-junctional voltage, or Vj, is defined as the membrane voltage of oocyte 2 minus the membrane voltage of oocyte 1.
The representative images show the sample Lj traces and the resulting normalized junctional conductance trans-junctional voltage relationship of UNC-9 homotypic gap junctions. Sample Lj traces and the resulting normalized Gj-Vj relationship of UNC-7b homotypic gap junctions are shown here. The results show that these two types of Gjs differ in the Vj-dependent Ij inactivation rate, Vj dependence, and the amount of residual Gj.While attempting the procedure, it's very important to make sure that the differential voltage electrode system does not have any air bubbles and its tip is aimed very close to the oocyte and the voltage and current electrodes have a resistance of approximately one microohm.
Our technique is easy to implement. It may be of particular interest to those labs that are newly interested in using Xenopus oocytes to study gap junctions.
